Effect of 3-subsitution of quinolinehydroxamic acids on selectivity of histone deacetylase isoforms

Abstract

A series of 3-subsituted quinolinehydroxamic acids has been synthesised and evaluated for their effect on human lung cancer cell line (A549), human colorectal cancer cell line (HCT116) and HDAC isoforms 1, 2, 6, and 8. The results indicated that substitution at C3 of quinoline is favoured for HDAC6 selectivity. Two compounds (25 and 26) were also found to be potent anti-proliferative compounds with IC₅₀ values ranging from 1.29 to 2.13 µM against A549 and HCT116 cells. These compounds displayed remarkable selectivity for HDAC6 over other HDAC isoforms with nanomolar IC₅₀ values. Western blot analysis revealed that compounds of this series activate apoptotic caspase pathway as indicated by cleavage of caspase 3, 8, and 9 and also increase phosphorylated H2AX thus inducing DNA double strand fragmentation in a concentration dependent manner. Flow cytometric analysis also displayed a dose dependent increase of cell population in sub G1 phase.

Keywords: HDAC; Quinoline; acrylamide; colon cancer; hydroxamic acid; lung cancer.

Graphical abstract

Figure 1.

Various examples of histone deacetylase inhibitors (1–5).

Figure 2.

Previously synthesised quinoline-containing HDAC inhibitors (6–13).

Figure 3.

Synthetic 3-substituted quinolinehydroxamic acids (14–29).

Scheme 1.

Synthetic Approaches to Compounds 14–29^a. ^aReagents and conditions: (a) NXS, acetic acid, 110 ^oC, 30–61%; (b) phenylboronic acid, Pd(OAc)₂, PPh₃, Na₂CO₃, THF, H₂O, MW, 90 ^oC, 90%; (c) CuI, 1,10-phenanthroline, Cs₂CO₃, MeOH, toluene, MW, 70 ^oC, 57%; (d) for 32–36: i. Fe powder, 1N HCl_(aq), EtOH (or MeOH), reflux; ii. methyl 4-formylbenzoate, NaBH(OAc)₃, DCM, 40 ^oC; for 37–47: 33, boronic acids, Pd(PPh₃)₄, TBAB, 2M K₂CO_3(aq), dioxane, reflux, 35–69%; (e) i. 1N LiOH_(aq), dioxane, rt; ii. NH₂OTHP, HBTU, DMF, TEA, rt; iii. 10% TFA_(aq), MeOH, rt, 28–55%.

Figure 4.

Effect of treatment with compound 17, 25, and 26 at three different doses (0.25 µM, 5 µM, and 10 µM) on cleavage of caspase 3, 8, and 9 and PARP. Increased expression of gamma H2AX, a phosporylated form of H2AX, indicates increased DNA double strand fragmentation of A549 cells treated for 48 h in a dose- dependent manner.

Figure 5.

(A) A549 cells were treated with or without 17, 25, and 26 (0.25, 0.5, 10 μM) for 48 h and were analysed by flow cytometry for cell cycle distribution. (B) After starvation for 24 h, A549 cells were then treated with compounds for the indicated time. After labelling with propidium iodide, DNA content was analysed by flow cytometry. (C) Quantification of cell population in Sub G1, G0/G1, S and G2/M phase. In A, B, and C, percent of cells = 100%.

Figure 6.

Docking of compound 17 (green) in the binding site of HDAC6 (PDB ID: 6CGP).